Purification and properties of RhaR, the positive regulator of the L-rhamnose operons of Escherichia coli.
Purification and properties of RhaR, the positive regulator of the L-rhamnose operons of Escherichia coli.
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DOI：
10.1016/0022-2836(90)90012-b
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发表时间：
1990-01
影响因子：
5.6
通讯作者：
J. Tobin;R. Schleif
中科院分区：
文献类型：
--
作者：
J. Tobin;R. Schleif
therhaRgene调节鼠李糖操纵子的四鼠李糖诱导启动子产生的mRNA水平，其产物已被超合成并通过双柱程序纯化。纯化后的蛋白是一个33 kDa的dna结合蛋白，与位于psrpromoter （hasandrhargenes的启动子）内的一个反向重复结构结合。测定了不同溶液条件下的平衡结合常数和动力学常数。该蛋白结合具有高亲和力，其结合对盐浓度和鼠李糖的存在敏感。RhaR接触的核苷酸和磷酸残基通过化学干扰法进行鉴定。所有的接触都是在DNA的一面进行的，对称模式与结合位点的反向重复序列相匹配。结合位点的一个不寻常的特性是，两个倒置重复序列的半位点彼此相隔17个碱基对的未接触DNA。如果17个碱基对通过插入完整的DNA而不是半完整的DNA来扩展，则会保留重要的结合。RhaR-DNA复合体明显弯曲，约为160°。
The product of therhaRgene, which regulates the level of mRNA produced from the fourl-rhamnose-inducible promoters of the rhamnose operon, has been hypersynthesized and purified by a two-column procedure. The purified protein is a 33 kDa DNA-binding protein that binds to an inverted repeat structure located within the psrpromoter, the promoter for therhaSandrhaRgenes. The equilibrium binding constants and kinetic constants have been determined under a variety of solution conditions. The protein binds with high affinity and its binding is sensitive to salt concentration and the presence ofl-rhamnose. The nucleotides and phosphate residues contacted by RhaR were identified by chemical interference assays. All of the contacts are made to one face of the DNA and the symmetrical pattern matches the inverted repeat sequence proposed for the binding site. An unusual property of the binding site is that the two half-sites of the inverted repeat are separated from one another by 17 base-pairs of uncontacted DNA. Significant binding is retained if the 17 base-pairs are extended by insertions of integral turns of DNA, but not by half-integral turns. The complex of RhaR-DNA appears to be sharply bent, approximately 160 °.